Expression of tyrosine hydroxylase (TH), the first and rate-limiting enzyme of catecholamine neurotransmitter biosynthesis, is limited to discrete sets of cells in the CNS and PNS. TH neurons of the CNS include the adrenergic and noradrenergic cells in the brainstem, dopaminergic cells of the midbrain and diencephalon (periventricular and hypothalamic nuclei), and the retinal amacrine cells and the dopaminergic cells in the olfactory bulb (OB). In the periphery, TH expression is largely limited to sympathetic ganglia and the adrenal medullary chromaffin cells. These peripheral TH cells are closely related: they derive from a common precursor that originates in the neural crest (Anderson and Axel, Cell, 47:1079-1090, 1986; Anderson et al., J. Neurosci., 11:3507-3519, 1991), and chromaffin cells from neonates can be transdifferentiated into cells that resemble sympathetic neurons by NGF (Aloe and Levi-Montalcini, Proc. Natl. Acad. Sci. USA, 76:1246-1250, 1979; Naujoks et al., Dev. Biol., 92:365-379, 1982; Doupe et al., J. Neurosci., 5:2119-2142, 1985). In contrast, CNS TH neurons arise from independent cell groups during neurogenesis, express TH at different times, and are functionally and anatomically distinct (Specht et al., J. Comp. Neurol., 199:233-253, 1981a; Bjorklund and Lindvail, Handbook of Chemical Neuroanatomy, Vol. 2, pp. 55-122, 1984). The mechanism responsible for TH expression in such disparate cell groups might be expected to rely on multiple regulatory elements, some that may be needed in all TH tissues and others that may mediate expression in a particular cell group.
Previous studies aimed at defining cis-acting DNA elements that direct TH expression have been of two sorts: those performed in vitro in cultured cells and those performed in vivo in transgenic mice. The in vitro work (Cambi et al., J. Neurochem., 53:1656-1659, 1989, the disclosure of which is incorporated by reference herein) compared expression of a chloramphenicol acetyl transferase (CAT) reporter gene under the transcriptional control of 5' flanking TH DNA in a TH-expressing pheochromocytoma (PC) cell line with that in a variety on non-TH-expressing lines. These experiments demonstrated that 212 base pairs (bp) of DNA upstream of the transcription start site were sufficient for TH transcription in PC cells and that sequences between -212 and -187 (where +1 is the start of the transcription) were required (Cambi et al., supra; Fung et al., J. Neurochem., 58:2044-2052, 1992). Further experiments using site-directed mutagenesis to pinpoint functional elements demonstrated that two sites known to bind transcription factors work together to direct appropriate expression in cultured cells (Yoon and Chikaraishi, Neuron, 9:55-67, 1992, the disclosure of which is incorporated by reference herein). One site is an API element (TGATTCA) at -205 that is bound by the members of the FosJun family of transcription factors (Curran and Franza, Cell, 55:395-397, 1988); the other contains an E-box motif (CAXXTG) at -194 that interacts with the transcription factors containing a helix-loop-helix motif like myc, MyoD, the products of achaete-scute, and E2A (Murre et al., Cell, 56:777-783, 1989; Blackwell and Weintraub, Science, 250:1104-1110, 1990). While these experiments provide fine resolution mapping of sites important for transcription in adrenal medullary PC cells, they give no information about tissue-specific expression in the neurons of the CNS or in sympathetic neurons, that is, in true neurons. In addition, the PC lines represent a limited range of developmental stages that may not reflect the mature state of differentiation. Due to the paucity of TH-expressing CNS cell lines, investigations of TH regulation in the CNS have used transgenic mice, where fine mapping is very difficult due to the time and expense involved in maintaining transgenic lines.
Although limited in number, previous experiments in which TH regulatory regions were used in transgenic mice have failed to demonstrate correct tissue-specific expression in all TH cells, although some TH cell groups were appropriately targeted. In a recent study, the human TH gene including the entire coding sequence, 25 kilobases (kb) of 5' flanking region, and 0.5 kb of 3' flanking region, was expressed in transgenic mice (Kaneda et al., Neuron, 6:583-594, 1991). The transgene was expressed in the brain and the adrenal, both TH-expressing tissues. However, primer extension analysis performed on RNA from various brain regions revealed inappropriate expression of human TH message in regions that lack TH-positive cell bodies; these regions included the frontal cortex, striatum, and the hippocampus. In fact, expression in the inappropriate areas appeared at least as strong as expression in TH-positive areas. This study did not examine expression in the sympathetic ganglia or in the main OB.
Similar results were obtained by Morgan and Sharp using sequences from the mouse TH gene linked to .beta.-galactosidase. They reported that transgenic mice carrying 3.5 kb of 5' flanking DNA demonstrated appropriate .beta.-galactosidase expression in the brainstem, midbrain, and adrenal, but ectopic reporter expression in other CNS regions, which may have been due to the site of integration (Morgan and Sharp, Abst. Soc. Neurosci., 17, 1991). Taken together, these studies indicate that none of the transgenic lineages so far examined have demonstrated completely correct tissue-specific expression. In all cases, some appropriate TH-positive cell groups do express the linked reporter, while others do not; in addition, ectopic CNS expression was observed in all three studies.
Immortalized cell lines of differentiated neuronal cell types, including those from the PNS and CNS, can be valuable research tools because they provide homogeneous sources of single cell types. They can be used to elucidate mechanisms of induction without the complicating presence of non-target cells, a situation impossible to achieve in vivo or in mixed primary cultures. They can also serve as sources for cell transplants of cell-specific molecules, as recipients for such molecules in gene transfer experiments or as sources for cell transplants into the nervous system. Neuronal cell lines from the central nervous system (CNS) are especially valuable since it is difficult to prepare our populations of primary neurons. In addition few differentiated cell lines originating from the CNS exist.
Neuronal cell lines have been generated by four methods: 1) from spontaneously arising or chemically induced tumors; 2) by fusion of neurons to neuroblastoma cells; 3) by retroviral infection of neural precursor cells; and 4) by the use of oncogenes driven by cell-specific promoters that direct tumorigenesis to defined neurons in transgenic mice. Although many cell lines have been derived from spontaneous or induced tumors, the majority are relatively undifferentiated (Spengler et al, In Vitro, 8:410, 1973; Schubert et al., Nature, 249:224-229, 1974; Waymire and Gilmer-Waymire, J. Neurochem., 31:693-698, 1978). Many have a mixed neuronal and glial phenotype characteristic of very mature cell types. The PC12 cell line (Greene and Tischler, Proc. Natl. Acad. Sci. USA, 73:2424-2428, 1976) and some mouse C-1300 subclones (Amano et al., Proc. Natl. Acad. Sci. USA, 69:258-263, 1972) do exhibit some differentiated properties. However, all of these are derived from the peripheral nervous system (PNS). Somatic cell hybrids offer the potential for obtaining immortalized cells with a well-differentiated phenotype, since mature neurons can theoretically be used as fusion partners. Unfortunately the success rate of such fusions is low and most researchers have relied on embryonic neuronal tissue to obtain hybrids which resemble mature neurons of varying degrees (Lee et al., J. Neurosci., 10:1779-1787, 1990; Choi et al., Brain Res., 552:67-76, 1991). An additional major drawback of somatic cell hybrid is the extent to which the neuroblastoma partner may influence the resulting hybrid, since the neuroblastoma parent is of PNS origin. This is of concern because hybrids contain additional chromosomes derived from the neuroblastoma parent and often lose those from the non-neuroblastoma parent. Recently, mesencephalic hybrid lines have been established by the fusion of embryonic mesencephalic cells with a neuroblastoma line (Choi et al., supra). Although these lines resemble mature midbrain neurons, they synthesize not only dopamine but also norepinephrine, which is surprising since mesencephalic cells are exclusively dopaminergic (Bjorklund and Lindvall, supra). Some of these problems can be circumvented by generating cell lines with retroviral or transgenic technologies. Retroviral infection of primary neuron cultures can result in neuronal cell lines capable of undergoing differentiation under appropriate conditions (Cepko, Ann. Rev. Neurosci., 12:47-65, 1989; Lendahl and McKay, Trends Neurosci., 13:132-137, 1990). Since the virus requires at least one round of cell-division in order to integrate into the host's genome, this approach has been largely limited to immortalization of cycling precursor cells (Fredericksen et al., Neuron, 1:439-448, 1988; Ryder et al., J. Neurobiol., 21:365-375, 1990). Transplantation experiments have shown that these precursor cells retain their plasticity when implanted into a developing brain, giving rise to cells appropriate for the site of implant (Renfranz et al., Cell, 66:713-729, 1991). Retroviral infection has thus been of greatest use for lineage analyses and other developmental studies.
The most direct approach to create cell lines of defined neuronal cell types utilizes tissue-specific promoter elements to direct oncogene expression in transgenic mice (Cory and Adams, Ann. Rev. Immunol., 6:25-48, 1988; Jenkins and Copeland, Important Advances in Oncology, Ed., 61-77, 1989, the disclosure of which is incorporated herein). In this way, very specific cells may be targeted. In the last few years this technique has led to the immortalization of gonadotropin releasing hormone (GnRH)-expressing and retinal amacrine neurons (Mellon et al., Neuron, 5:1-10, 1990; Hammang et al., Neuron, 4:775-782, 1990; Messing et al., 1991, Soc. for Neurosci., abstract #21.11), as well as differentiated cells of many non-neuronal tissues (Hanahan, Nature, 315:115-122, 1989). Since the target cell destined for transformation is determined by the regulatory promoter elements used, in theory, tumors can be induced in any cell type for which specific regulatory elements are available. For the most part, tumors have been observed in the appropriate tissues, although ectopic expression does occur (Cory and Adams, supra; Behringer et al., Proc. Natl. Acad. Sci. USA, 85:2648-2652, 1988).
Thus, it would be desirable to provide a full repertoire of TH-expressing cells which express a target gene under the control of a regulatory region which permits correct expression in such cells. Similarly, it would be desirable to immortalize such cell lines under the control of a regulatory region which drives an oncogene and which permits correct expression.